Auricular cartilage regeneration using different types of mesenchymal stem cells in rabbits

Background Cartilaginous disorders comprise a wide range of diseases that affect normal joint movement, ear and nose shape; and they have great social and economic impact. Mesenchymal stem cells (MSCs) provide a promising regeneration alternative for treatment of degenerative cartilaginous disorders. This study aimed to compare therapeutic potential of different types of laser activated MSCs to promote auricular cartilage regeneration. Twelve adult rabbit allocated equally in four groups, all animals received a surgical mid auricular cartilage defect in one ear; Group I (Positive control) injected sub-perichondrially with phosphate-buffered saline (PBS), Group II (ADMSC-transplanted group) injected adipose-derived MSCs (ADMSCs), Group III (BMMSCs-transplanted group) received bone marrow-derived MSCs (BMMSCs), and Group IV (EMSC-transplanted group) received ear MSCs (EMSCs) in the defected ear. The auricular defect was analyzed morphologically, histopathologically and immunohistochemically after 4 weeks. In addition, a quantitative real-time polymerase chain reaction was used to examine expression of the collagen type II (Col II) and aggrecan as cartilage growth factors. Results The auricles of all treatments appeared completely healed with smooth surfaces and similar tissue color. Histopathologically, defective areas of control positive group, ADMSCs and EMSCs treated groups experienced a small area of immature cartilage. While BMMSCs treated group exhibited typical features of new cartilage formation with mature chondrocytes inside their lacunae and dense extracellular matrix (ECM). In addition, BMMSC treated group showed a positive reaction to Masson’s trichrome and orcein stains. In contrary, control positive, ADMSC and EMSC groups revealed faint staining with Masson’s trichrome and Orcein. Immunohistochemically, there was an intense positive S100 expression in BMMSCs (with a significant increase of area percentage + 21.89 (P < 0.05), a moderate reaction in EMSCs (with an area percentage + 17.97, and a mild reaction in the control group and ADMSCs (area percentages + 8.02 and + 11.37, respectively). The expression of relative col II and aggrecan was substantially highest in BMMSCs (± 0.91 and ± 0.89, respectively). While, Control positive, ADMSCs and EMSCs groups recorded (± 0.41: ± 0.21, ± 0.6: ± 0.44, ± 0.61: ± 0.63) respectively. Conclusion BMMSCs showed the highest chondrogenic potential compared to ADMSCs and EMSCs and should be considered the first choice in treatment of cartilaginous degenerative disorders.

restorative potential. This evaluation was done using the most accurate and confirmative anatomical, histological, and immunohistochemical studies. Moreover, biochemical diagnostic studies were performed for collagen type II (col II), aggrecan and TGF-b1 expression through a distinctive molecular analysis.

Animals
Six-month-old adult New Zealand white rabbits (Oryctolagus cuniculus), both male and female, with a weight range of 2.5-4 kg, were purchased and raised in a pathogen-free environment (at the Department of Anatomy and Embryology using the Close Battery System). The animal study protocol was approved by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Cairo University (Giza, Egypt; Vet. CU. IACUC reference no. VetCU8032022406). Twelve New Zealand rabbits were numbered and divided equally (three rabbits per group) into four groups, and auricular cartilage defect was induced in all of these groups.

Induction of cartilage defects
Both right and left ears of the 12 rabbits were utilized, and grouped in: positive control, ADMSCs-treated, BMMSCs-treated, and EMSCs-treated groups. All rabbits were anesthetized by intravenous ear vein injection with 2% xylazine (1-3 mg/kg) (Bayer, Leverkusen, Germany) and ketamine (2 mg/kg) (Virbac, Carros, France) In each rabbit from all groups, 2 × 2 cm cartilage plate was removed from the midportion of each auricle.

Isolation of rabbit ADMSCs
Under general anesthesia induced by intravenous administration of 2% xylazine (1-3 mg/kg) and ketamine (2 mg/ kg), an incision was made in the skin and subcutaneous tissue of the inguinal region. Subcutaneous fat (10 g) was extracted from bilateral inguinal adipose tissue and washed with PBS to remove excess blood. Adipose tissue was minced into small pieces using blades and treated with 1 mL of 0.075% collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) in a shaking water bath for 60 min at 37 °C and a 5% CO 2 incubator. Enzyme activity was neutralized with 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle's Medium (DMEM), and the sample was centrifuged at 4000 rpm for 10 min to form a pellet. To remove cellular debris, the pellet was filtered through a 100 µm nylon mesh and centrifuged for 10 min at 3000 rpm, and the supernatant was carefully aspirated to obtain cell pellets composed of stromal vascular fraction cells.

Isolation of rabbit BMMSCs
Bone marrow for BMMSCs isolation was collected from the whole femur or tibia of the rabbits after euthanasia. Two rabbits were sloughed, and the peripheral muscle tissue was removed. The femur and tibia were washed in alcohol and rinsed twice with PBS containing 1% penicillin/streptomycin. Bone marrow was flushed out with 5-mL DMEM in Falcon containing 0.1-mL heparin (3000 U/mL) or 1-mL acid citrate dextrose. Cells were isolated by Ficoll-Paque density gradient centrifugation, 0.7-mL Ficoll-Paque added and centrifuged at 1000 rpm for 10 min, after which the supernatant was discarded. The cell pellet was resuspended in 2-ml red blood cell (RBC) lysis buffer containing 0.83% ammonium chloride (to destroy RBCs) and centrifuged at 3000 rpm for 10 min.

Isolation of rabbit EMSCs
Under anesthesia, a medium ear Section (2 cm diameter) with no significant vessels was punctured. After punching, the wound was cleaned with oxytetracycline spray. The skin outer layers and connective tissue were removed, and the remaining cartilage was washed in PBS before being cut with blades. The cartilage was processed for 3 h at 37 °C in a 5% CO 2 incubator with 3 mL of 0.075% collagenase type II [26]. To obtain a pellet, enzyme activity was neutralized with DMEM, and the sample was centrifuged at 4000 rpm for 10 min.

Tissue culture of the three different MSCs
The pellet was resuspended in 10-ml PBS, and cells were cultured in a 75-cm 2 culture flask that contained 12-mL DMEM high glucose with 10% FBS and 100 mg/mL penicillin/streptomycin, and cells were incubated at 37 °C with 5% humid CO 2 . After 48 h, the medium was changed to remove nonadherent cells, and adherent cells were cultured until they reached 80-90% confluence. The culture medium was changed every 3 days, and cells proliferated until the third passage. After the adherent cell monolayer had attained confluence, cells were trypsinized with 2 mL of 0.05% trypsin-EDTA and incubated for 2 min. Excess trypsin was then removed by adding DMEM, followed by centrifugation and subculture again. For the experiments, third or fourth passage of stem cells was used.

MSC low laser activation
After trypsinization, the pellet that contains stem cells was suspended in PBS and activated using a low-level pulsed red laser light (625 nm) at room temperature for 10 min from distance of 7 cm with peak irradiance intensity of 80 mW/cm 2 [29].

Subperichondrial injection of stem cells
After trypsinization, the pellet that contains stem cells was resuspended in PBS and activated using a low-level laser for 10 min. Approximately 1 mL purified stem cells with PBS were then injected at a concentration of 2 × 10 7 cells/mL at the excision site's margin with a 29-gauze needle in each group according to the type of stem cells on PODs 0, 2, and 4. In the positive control group, 1mLPBS was injected into the defect site.

MSC proliferation assay
The growth of MSC of different sources was measured using Trypan Blue (Thermo Fisher Scientific) cell viability assay at 1, 3, 5, 7, and 9 days after the initial MSC seeding. Using a 48-well culture plate, 2,000 MSC were plated in triplicate for each time point. At each time point, Trypan Blue was added to each well and incubated at 37 °C for 1 min. The cells number was counted using inverted bright field microscope (EXI-600 is ACCU-SCOPE's, USA).

Stem cell characterization
Flow cytometry The phenotypes of stem cells were studied using flow cytometry. Following the second passage, stem cells were harvested. Cells were treated with a 10% trypsin EDTA solution for 5-10 min in the incubator, followed by a wash. The cell pellet was then incubated for one hour with 1% bovine serum albumin containing primary antibodies against the following surface markers: CD105, CD73, CD90, CD44, CD45 CD34, CD19.The cells were then incubated for 30 min with the secondary antibody before immunophenotyping using a fluorescenceactivated cell sorting cell analyzer (CytoFLEX, Beckman Coulter, United States). No isotype control antibody is used.
Gating: forward and side scatter (FSC, SSC) gating is used for identifying the cell population and exclude the debris.
The meaning of light blue color (cells negative to respective surface marker), Red cell indicate positive expression to respective surface marker. stem cells differentiation The Mesenchymal Stem Cells were analyzed for their capacity to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Cells from the third passage were seeded in 6-well plates at a density of 20,000 cells/cm 2 and differentiated in vitro using adipogenesis kits (A1007001; Gibco StemPro, USA) or osteogenesis kits (A1007201; Gibco StemPro) for 3 weeks according to the manufacturer's instructions. The media refreshed every 3 days. While chondrogenic differentiation, the cells were differentiated with a Chondrogenesis Kit (A1007101; Gibco StemPro) for 3 weeks. Cells differentiated using an adipogenesis or osteogenesis kit were stained with 0.5% Oil Red O or Alizarin Red S for 1 h, respectively. Oil Red O staining, a marker for intracellular lipid accumulation, and Alizarin Red S staining, a marker for extracellular matrix calcification While, Safranin O stain was used to stimulate chondrogenic development [30].

Morphological evaluation of cartilage regeneration
Inspection of both ears of each animal was continued until the auricular defect was completely closed. Photographs were taken with a digital camera (Nikon D40, Nikon, Japan) immediately on PODs 0, 3, 7, 14, 21, and 28.

Microscopical evaluation
Samples from all groups were carefully dissected, fixed in 10% neutral buffered formalin (NBF) for 48 h, washed, and embedded in ascending grades of ethanol for dehydration. Tissue sections were cleared in xylene, embedded in paraffin wax, and sectioned at 3-5 μm thickness for histopathological and immunohistochemical examinations.

Histopathological examination
The prepared deparaffinized Sections (3-5 μm) were eventually stained with hematoxylin and eosin (H&E), Masson's trichrome, and toluidine blue stains [31], in addition to orcein stain that detects elastic fibers [32]. The stained slides were examined using a light microscope (Leica DM500). The images were captured by a camera (Leica ICC50 HD) attached to the microscope and finally examined by an image analysis software [Leica Microsystems, LAS version 3.8.0 (Build: 878); Leica image analyzer computer system] at the Cytology and Histology Department, Faculty of Veterinary Medicine, Cairo University.

Immunohistochemical examination for S100
Deparaffinized sections (thickness, 4 μm) from all groups were prepared for S100 immunohistochemical examination according to [33]. S100 immunostaining was quantified as area percentage [using ImageJ software (NIH, Bethesda, MD, USA)] in randomly selected high-power microscopic fields from different sections for each group. Regardless of the severity of staining, areas displaying positive S100 (brown color) were chosen for estimation. Each specimen's mean value and standard error of the mean were calculated and statistically analyzed. S100 immunostaining was measured as area % in a standard measuring frame in representative five fields in each group using 400 × magnification via light microscope transferred to the screen. The areas showing positive brown immunostaining were chosen for evaluation regardless the intensity of staining. Each specimen's mean value and standard error mean (SEM) were calculated and statistically analyzed. The data was analyzed using one-way analysis of variance (ANOVA) by SPSS version 17.0 software (IBM, USA) to assess the significance of the mean between the groups, followed by an LSD post hoc test. Statistical significance was described as a P-value less than 0.05.

Statistical analysis
Data was analyzed using a one -way analysis of variance (ANOVA) by SPSS version 17.0 software (IBM, USA) to assess the significance of the mean between the groups, followed by a least significant difference (LSD) Fischer post hoc test. Statistical significance was described as P˂0.05. values are presented as mean ± SEM (n = 3 rabbits/group). Different superscript letters indicate a significant difference at p ≤ 0.05.

MSCs proliferation assay
To show the growth of MSCs over time, the increased cell count throughout the designated time points of 1, 3, 5, 7, and 9 days were detected as the values of 6133, 8545, 8448, 11,623, and 13,047, respectively (Fig. 1).

Stem cell characterization Flow cytometry
Flow cytometric characterization analysis of rabbit stem cells in Fig. 2A (Fig. 3). Osteogenic differentiation was confirmed by the deposition of Alizarin Red S-stained mineralized matrix. Calcification appeared as red regions within the cell monolayer (Fig. 3). Chondrogenic differentiation was confirmed by Safranin O stain that was used to stimulate chondrogenic development (Fig. 3).

Gross anatomical findings
During the trial, no auricles indicated infection, inflammation, or graft rejection symptoms. The defect in the control group remained narrow, with no cartilage growth within the damage (Fig. 4D). After 4 weeks of MSC injection, gross observation revealed that the cartilaginous defect was entirely healed by chondrocytes with a smooth surface and similar color to the surrounding tissue in the experimental groups ( Fig. 4A-C).

Microscopic findings Histopathological evaluation
H&E-stained sections of the untreated (control) group revealed a defective area at the location of the surgically removed auricular cartilage. Except for a small island of cartilage, no new cartilaginous tissue was observed (Fig. 5A, B). ADMSCs injection resulted in the formation of small immature cartilaginous tissue at the defective edges that appeared light purple in H&E staining, distinguishing it from the dark purple mature original cartilage (Fig. 5C).
Interestingly, defective areas that received BMMSCs had the greatest area of new cartilage formation that exhibited typical features, namely, mature chondrocytes inside their obvious lacunae and dense ECM (Fig. 5D, E). This newly formed cartilage was surrounded by collagenous tissue with differentiated chondrocytes (Fig. 5E, F). Moreover, EMSCs showed the formation of new immature cartilage arranged in continuity with the native cartilage remnant to fill the cartilage defect (Fig. 5G). This formed cartilage revealed a light purple matrix surrounding chondrocytes that appeared not evenly distributed (Fig. 5H, I). The defective area refers to the site of the surgically created auricular cartilage defect "as shown in Fig. 4 by double arrow line".
In contrast, Masson's trichrome staining of the control group revealed a pale bluish differentiated tissue at the defective edges of the original cartilage (Fig. 6A). This tissue showed immature chondrocytes (Fig. 6B) surrounded by elastic fibers that appeared brown when stained with orcein (Fig. 6C). Moreover, the small formed immature cartilage at the defective edges treated with ADMSCs showed a faint reaction to Masson's trichrome (Fig. 6D) and orcein (Fig. 6E) stains.
Defective areas treated with BMMSCs showed a positive reaction of the newly formed cartilage to Masson's trichrome stain (Fig. 6F) and orcein stain that showed dark brown branching pericellular elastic fibers. However, faint brown coloration was observed in the surrounding tissue, indicating the predominance of collagen  (Fig. 6G). In addition, the EMSC-treated group revealed a pale blue, new cartilage after staining with Masson's trichrome stain (Fig. 6H). This cartilage showed no mature branching strands of pericellular elastic fibers; instead, faint brown coloration was noticed at the edges of the defective area of the original cartilage that exhibited dark brown mature elastic fibers (Fig. 6I).
Moreover, the microscopic structure of the examined groups was investigated using a toluidine blue stain, in which aggrecan exhibit purple color (Fig. 7). The control group revealed the light blue color of the newly formed tissue matrix (Fig. 7A). The ADMSC-treated tissue revealed a faint bluish tint at the defective edges (Fig. 7B). Interestingly, BMMSCs-treated (Fig. 7C, D) and EMSCstreated (Fig. 7E, F) groups exhibited a deep purple coloration of the newly formed cartilage at the defective edges. This cartilaginous tissue could be distinguished from the original cartilage by small-sized chondrocytes that appeared not evenly distributed.

Immunohistochemical evaluation
Tissues from the control and ADMSCs-treated groups showed mild reaction to S100 with area percentages of 8.02 and 11.37, respectively. Moderate immune reactivity was observed in EMSC treatment, with an increase in area percentage of 17.97. Interestingly, BMMSCs revealed an intense positive expression of S100, with a significant increase in area percentage to 21.89 (Fig. 8).
The control negative is healthy and normal tissue; thus all our target genes are downregulated in other groups because they all suffered from auricular injury. We performed one-way ANOVA to analyze our data. Different superscript letters indicate significant differences between each other P < 0.05.
Expression of col II and aggrecan was substantially higher in BMMSCs than in the other groups. The stem cell-treated samples showed significantly higher col II and aggrecan gene expression than the positive control group.

Discussion
Tissue and organ regeneration based on stem cell therapy provides alternative medical solutions for many affections. MSCs exhibit adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic potential and provide a cell source for cartilage regeneration [35,36]. This study was designed to evaluate the regenerative potential of different types of MSCs (Table 1).
This investigation agreed with [37], who revealed that the defects of ear cartilage have poor support for vascularization. Therefore, the regenerative capacity for selfrepairing and regeneration would have less potential. However, anatomically, the cartilage is an essential target for tissue engineering due to its immune prerogative and the presence of dense ECM surrounding the lymphatic tissue found invincible to leukocytes. In our study that was able to determine the regeneration effects of BMMSCs, ADMSCs, and EMSCs on auricular cartilage defect, although [3,38] proved that (ADMSCs) generated from adipose tissue might multiply and develop into chondrocytes.
In available literatures were unclear whether BMMSCs, ADMSCs, or EMSCs injected into an auricular cartilage defect were surgically divided, differentiated, and gave rise to freshly formed chondrocytes and their orchestrated regeneration by stimulating bioactive factors. In this study, there was adequate time to show auricular cartilage defect regeneration and wound healing with notable differences between each kind of MSCs.
Although ADMSCs are widely available and relatively painless to be collected in large quantities, they are regarded as an additional source of MSCs for tissue repair and engineering [35,36]. This study added that BMMSCs are a representative cell source to improve wound healing in various ways and develop into effector cells engaged in angiogenesis, ECM formation, wound contraction, reepithelialization, and matrix secretion. This result was also supported by [20,39].
Oh, Park [3] and Khalilifar, Eslaminejad [26] illustrated the accelerated repair of auricular cartilage defects in ADMSCs-treated rabbits. Compared to control, auricular cartilage regeneration in these rabbits was morphologically enhanced, and healing improvement revealed that cartilaginous lesions were entirely restored by chondrocytes with a smooth surface and identical color to the surrounding tissue, these results were similar with our observations in the present study.
Based on comparative in vitro investigation report [26], ADMSCs had the highest proliferation rate with the least propensity for chondrogenic differentiation. Moreover, BMMSC-and EMSC-seeded scaffolds revealed an efficient improvement of cartilage defect 4 weeks after transplantation. This investigation agreed with our study, that in which the ADMSCs revealed higher proliferation rate while BMMSCs showed higher cartilage differentiation potential.
Compared to other MSCs types, defective auricular areas that received BMMSCs had the greatest area of new cartilage formation histopathologically. BMMSCs exhibited typical cartilage features: mature chondrocytes inside their obvious lacunae and dense ECM confirmed by Masson's trichrome and orcein stains that showed positive reaction to Masson's trichrome and dark brown branching pericellular elastic fibers with orcein. This finding agreed with [15], who noted that the defective area was completely repaired by mature, evenly distributed chondrocytes with obvious lacunae after 18 weeks of BMMSCs treatment, making them good candidates for cartilage defect regeneration. This was in contrast to Khalilifar,Eslaminejad [26], who recorded that EMSCs were the only appropriate option for promoting cartilage reconstruction, as shown in histological assessments and gene expression.
In contrast to [3,40], who noted significant regenerative effects of ADMSCs after 1 and 2 months, respectively, our results of the H&E-stained sections revealed small immature cartilaginous tissue formation only at the defective edges of the original cartilage without any new cartilage formation in the defect area after 1 month of ADMSCs injection. Faint reaction to Masson's trichrome and Orcein stains supported the H&E findings. Moreover, Bahrani, Razmkhah [38] did not observe the formation of small new immature cartilage islands until 3 months after injection with ADMSCs, but they detected the formation of a mature cartilage plate in the gap after 5 months. Well-formed mature cartilaginous plate that completely filled the gap was formed 6 months after injection.
Based on our investigations, the comparison between different types of MSCs showed higher proliferation rate of ADMSCs followed by BMMSCs then EMSCs. Results of Khalilifar, Eslaminejad [26] support our observation while EMSCs came in the second order after ADMSCs. In this study we performed a subchondral injection as a route of administration of MSCs, in contrast Khalilifar, Eslaminejad [26] applied MSCs on scaffold directly on defected cartilage. On macroscopic evaluation, all treated Fig. 9 Gene expression analysis of ADMSCs, EMSCs and BMMSCs. Bar chart representing the transcript level of (Collagen Type II, Aggrecan, and TGF-b1) in different groups at 4 weeks post treatment. values are presented as mean ± SEM (n = 3 rabbits/group). Data were calculated by 2− ∆∆CT method, using (actin-β) as internal control, and then normalized to the control negative value which was set to be 1. The control negative is healthy and normal tissue; thus all our target genes are downregulated in other groups because they all suffered from auricular injury. We performed one-way ANOVA to analyze our data. Different superscript letters indicate significant differences between each other P < 0.05 groups looks similar in color to surrounding tissue with smooth surface, but Khalilifar, Eslaminejad [26] stated a significant difference between BMMSCs, EMSCs and that of ADMSCs. Khalilifar, Eslaminejad [26] recommended the use of EMSC for their highest chondrogenic differentiation and best histological findings. On the other hand, our results on histological and gene expression levels showed preference of BMMSCs over EMSCs.
Bahrani, Razmkhah [38] performed the study on ADMSCs which didn't show any obvious enhancement on macro and microscopic level after 1 month of treatment. The enhancement of cartilaginous repair was detected starting from the 3rd month with complete healing after 6 months. In contrast, our study was performed to compare three different types of MSCs at short term treatment schedule which indicated that BMMSCs are the preferred type of MSCs in treatment of cartilaginous defect on short term (1 months of injury).
In contrast, S100 proteins play a major role in many intracellular and extracellular biological activities. Intracellular functions include cell differentiation and survival [41]. This study immunohistochemically evaluated its expression in differentiated chondroblasts. Our finding revealed that BMMSCs had a strong positive expression of S100, with a significant increase in area percentage (21.89), compared to control (8.02). A moderate reaction was obtained in EMSCs, with an area percentage of 17.97, whereas mild expression (area percentage of 11.37) was observed in the ADMSCs-treated group. These findings contradicted [3], who stated that ADMSCs transformed to a chondrogenic pattern with increased expression of S100 protein. Their result did not simulate comparative findings in this study.
Cartilage contains up to 10% proteoglycan, mainly composed of a large aggregated chondroitin sulfate, proteoglycan aggrecan [42]. Aggrecan plays a crucial role in tensional connective tissues [43]. The interaction between aggrecan and hyaluronan forms a macroaggregate structure physically confined in the col II network of cartilage tissue [44]. Chondrocytes expressing high levels of aggrecan and link proteins are maintained within the matrix network and survive in suspension cultures [45].
Many tissues, including skin, skeletal muscle, the gastrointestinal tract, the liver, and bone seem to depend on growth factors for wound healing and regeneration [46]. TGF-b1 (transforming growth factor) plays a central role in stem cell and tissue homeostasis. In the current study we report upregulation of the TGF-b1, or transforming growth factor during ear cartilage wound healing. The tendons and cartilage injury caused suppression of the TGF-b1 expression [47].
Transforming Growth Factor (TGF-b1) is a pro-migratory factor that supports wound healing, modify the extracellular matrix and promote cell proliferation and angiogenesis [48]. TGF-b1 mediates the suitable migration of MSCs to appropriately integrate into the remodeling tissue [49].
Oh, Park [3] concluded that ADMSCs had a regenerative effect on auricular cartilage defect in rabbits with higher col II expression, whereas [26] asserted that EMSCs had the highest expression of aggrecan in articular cartilage regeneration. However, our investigations proved that the relative expression of col II and aggrecan was significantly higher in BMMSCs (0.91 for col II and 0.89 for aggrecan) than other MSCs.

Conclusion
In this study, BMMSCs had the highest proliferation rate and chondrogenic potential compared to ADM-SCs and EMSCs, as shown in histological assessments, with better reactivity of the S-100 protein and higher production of col II, aggrecan and TGF-b1, which could be of superior value over ADMSCs and EMSCs for the regeneration of the cartilaginous defects.